Outer membrane proteins of Escherichia coli. I. Effect of preparative conditions on the migration of protein in polyacrylamide gels

Outer membrane protein of Escherichia coli prepared for polyacrylamide gel electrophoresis by solubilization of the membrane in an organic solvent followed by dialysis into sodium dodecyl sulfate (SDS) solution or by solublization of the membrane directly in SDS solution followed by dialysis into a SDS-urea solution and brief heating at 100 °C resulted in a simple polypeptide profile on SDS-containing gels. This polypeptide pattern was characterized by a single major protein band migrating with an apparent molecular weight of about 42,000 daltons which accounted for about 70% of the total protein on the gel. However, if the outer membrane protein is dissolved in SDS solution without urea and heated at 70 °C, major bands are observed in three regions of the gel: A broad band or group of bands near the top of the gel with an apparent molecular weight of much greater than 42,000 daltona (peak A), a second band with the same mobility as the 42,000-dalton band in boiled samples (peak B), and a third, faster-migrating band with an apparent molecular weight of less than 42,000 daltons (peak C).Elution of protein from A or C followed by heating at 100 °C converts this protein to a form migrating with peak B. If the outer-membrane protein is dissolved in SDS solution at 37 °C with no further heating and applied to gels, peak B dissappears completely and A and C increase. These can be partially converted to peak B by urea treatment. Protein from peaks A and C was isolated by chromatography on Sephadex in the presence of SDS, and the intrinsic viscosity of this protein was measured before and after boiling. The intrinsic viscosity of protein from peak A was 35 cc/g both before and after boiling, while the intrinsic viscosity of protein from peak C was 28 cc/g before boiling and 35 cc/g after boiling. These results are best explained by assuming that the protein in peak A represents aggregates of a 42,000-dalton species which are dissociated by boiling or solvent treatment and that the protein in peak C represents a monomeric form of the 42,000-dalton protein which is not fully reacted with SDS and which is converted to the “rigid rod” conformation characteristic of protein-SDS complexes only upon boiling or solvent treatment.